Delivery of pharmaceutical and therapeutic agents is often severely limited by the chemical or physical barriers of the body. For example, oral administration of therapeutics is the route of choice. However, the extreme pH values and powerful digestive enzymes in the digestive system often destroy a therapeutic agent before it can get into the bloodstream and have any beneficial effect. Therapeutic agents such as biologically active peptides and proteins (e.g., insulin) are generally perceived to be unsuitable for oral administration because they are rapidly destroyed in the digestive system by acid hydrolysis and/or by proteolytic enzymes.
A great deal of research has been devoted to developing effective oral drug delivery methods and systems for these vulnerable therapeutic agents. For example, workers have attempted to co-administer the agent with an adjuvant such as a resorcinol containing a non-ionic surfactant like polyoxyethylene oleyl ether or n-hexadecyl polyethylene ether to increase the permeability of the intestinal walls. Other workers have attempted to co-administer therapeutic agents with enzyme inhibitors like pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol to avoid enzymatic degradation.
However, these approaches have limited applicability because the added reagents are toxic, interact negatively with the therapeutic agent, or fail to adequately protect the therapeutic agent or the uptake and absorption of the therapeutic agent.
Liposomes have also been used as drug delivery agents. They provide a layer of lipid around the encapsulated pharmacological agent. For example, the use of liposomes containing heparin is disclosed in U.S. Pat. No. 4,239,754. Several studies have been reported on the use of liposomes with insulin; e.g., Patel et al. (1976) FEBS Letters Vol. 62, page 60 and Hashimoto et al. (1979) Endocrinol. Japan, Vol. 26, page 337.
However, problems also exist for delivery with liposomes. Liposome suffer from poor stability, inadequate shelf life, small cargo loads (molecular weights generally less than 30,000), difficulty in manufacturing and adverse interactions with cargoes.
Artificial amino acid polymers or proteinoids that are form into microspheres have been described for encapsulating pharmaceuticals. See, e.g., Ma et al., 1994; Santiago et al., 1993; Madham Kumar and Pandurango Rao, 1998. Proteinoid microspheres can be formed by the thermal condensation of amino acids. The reaction joins carboxyl and amino groups that are part of an amino acid side chain or that are in the α position. A generalized scheme for a proteinoid thermal condensation reaction is illustrated below.
The carboxylate can be donated from either an amino acid sidechain (Asp or Glu) or from the α carboxylate. Similarly, the free amino group can be donated from either an amino acid sidechain (Arg, Lys, Asn, or Gln) or from the α amino group. It is relatively straightforward to produce proteinoids that are either acidic or basic via this simple thermal condensation reaction.
Such thermal condensation of simple amino acid mixtures was first described in the late 1950's by Harada and Fox (1958, 1960). This early work focused on linear polymers as model protein systems. It was not until 1964 that scientists realized that the thermal condensation of amino acids could result in the formation of spherical particles (Fox 1964). Proteinoid microspheres made in this manner can vary in size from 1 to 10 microns and some of them are hollow.
A proteinoid can be swelled in aqueous solution at moderate temperatures (usually around 50° C.) forming a structure known as a microsphere (see, e.g. Brooke and Fox, 1977). Several workers have formulated experimental models for ‘communication’ between proteinoid microspheres (Hsu et al., 1971 and Hsu and Fox, 1976) that involves conjugation and fusion of individual proteinoid microspheres. Considering the abiotic conditions on earth millions of years ago and the conditions leading to truly living complex assemblies, such workers have suggested that proteinoid microspheres could be involved in a natural transition from cell-like structures to actual cells.
Researchers have searched for enzymatic activity in proteinoid microsphere preparations. Most of the early work in this area was motivated by an interest in supporting an evolutionary role for proteinoid microspheres. Reports exist on the catalyzed formation of small linear peptides (Nakashima and Fox, 1980 and 1981; Fox and Nakashima, 1980), the activation of glycine (Ryan and Fox, 1973), and the formation of oligonucleotides (Jungck and Fox, 1973). Other reports describe proteinoid microsphere-mediated catalysis (Masinovsky, 1995), and the use of porphyrin complexed proteinoid microspheres as photosensitizers (Masinovsky et al., 1989). Yet a review of the literature reveals that proteinoid microspheres are actually devoid of what is generally defined as true enzymatic activity.
Once formed, thermal proteinoid microspheres are stable (Muller-Herold and Nickel, 1994; Syren, et al., 1985). In addition, thermal proteinoids are relatively easy to synthesize and characterize (Fox and Nakashima, 1966; Phillips and Melius, 1974; Luque-Romero et al., 1986; Kokufuta et al., 1983). A theoretical foundation for the assembly and self assembly of proteinoid microspheres has been formulated (Matsuno, 1981; Matsuno, 1981b). Some workers have even documented membrane-like electrical potential in proteinoid microspheres (Matsuno, 1984; Przybylski, 1985; Przbylski et al., 1982; Ishima, et al., 1981). Another report by Brooke and Fox (1977) describes the complex nature of proteinoid microsphere compartmentalization, including the responses of internal compartments to changes in temperature and pH.
U.S. Pat. No. 4,925,673 (the '673 patent) describes thermal proteinoid microspheres as well as methods for their preparation and use. However, the physicochemical properties of the proteinoid microspheres described in the '673 patent are not optimal. The light sensitivity, shelf life and the solubility in various portions of the gastrointestinal tract are limited. Additionally, there is a need for microspheres that can encapsulate a broader range of active agents such as lipophilic drugs.
Moreover, the method for making proteinoid microspheres described in the '673 patent provides a complex mixture of high molecular weight and low molecular weight peptide-like polymers which are difficult to separate. Only a small amount of the low molecular weight proteinoids form microspheres. Hence, an improved method of preparing of the proteinoids is also desired.
Accordingly, there is a need in the art for improved proteinoid microspheres as well as improved methods for their preparation.